WO2003018626A2

WO2003018626A2 - Novel genetic products from ashbya gossypii, associated with the structure of the cell wall or the cytoskeleton

Info

Publication number: WO2003018626A2
Application number: PCT/EP2002/009355
Authority: WO
Inventors: Marvin Karos; Henning ALTHÖFER; Burkhard Kröger; Jose L. Revuelta Doval
Original assignee: Basf Aktiengesellschaft
Priority date: 2001-08-22
Filing date: 2002-08-21
Publication date: 2003-03-06
Also published as: CA2456828A1; CN1543475A; JP2005522984A; US20050221460A1; KR20040029000A; EP1421110A2; WO2003018626A3; AU2002327839A1

Abstract

The invention relates to novel polynucleotides from Ashbya gossypii; oligonucleotides hybridised therewith; expression cassettes and vectors, containing said polynucleotide; microorganism transformed therewith; polypeptides coded by said polynucleotides and the use of the novel polypeptides and polynucleotides as targets for modulation of the cell wall or cytoskeletion construction and in particular for improving the production of vitamin B2 in microorganisms of the species Ashbya.

Description

description

New gene products from Ashbya gossypu, which are associated with the structure of the cell wall or the cytoskeleton.

The present invention relates to novel polynucleotides from Ashbya gossypu; oligonucleotides hybridizing therewith; Expression cassettes and vectors containing these polynucleotides; microorganisms transformed therewith; polypeptides encoded by these polynucleotides; and the use of the new polypeptides and polynucleotides as targets for modulating the cell wall or cytoskeleton properties and in particular for improving vitamin B2 production in microorganisms of the genus Ashbya.

Vitamin B2 (riboflavin, lactoflavin) is an alkali and light sensitive vitamin that fluoresces yellow-green in solution. Vitamin B2 deficiency can lead to ectoderm damage, especially clouding of the lens, keratitis, comea vascularization, neurovegetative and urogenital disorders. Vitamin B2 is the precursor for the biological hydrogen transfer molecules FAD and FMN, which are important in addition to NAD ^* and NADP ⁺ . These are formed from vitamin B2 by phosphorylation (FMN) and subsequent adenylation (FAD).

Vitamin B2 is synthesized in plants, yeasts and many microorganisms from GTP and ribulose-5-phosphate. The pathway begins with the opening of the imidazole ring from GTP and the cleavage of a phosphate residue. 5-Amino-6-ribitylamino-2,4-pyrimidinone is formed by deamination, reduction and elimination of the remaining phosphate. The reaction of this compound with 3,4-dihydroxy-2-butanone-4-phosphate leads to the bicyclic molecule 6,7-dimethyl-8-ribityllumazine. This compound is converted into the tricyclic compound riboflavin by dismutation, in which a 4-carbon unit is transferred.

Vitamin B2 is found in many vegetables and meat, less in cereal products. An adult's daily vitamin B2 requirement is around 1.4 to 2 mg. The main breakdown product of the FMN and FAD coenzymes in humans is again riboflavin, which is excreted as such.

Vitamin B2 is therefore an important nutritional supplement for humans and animals. There is therefore a desire to make vitamin B2 accessible on a technical scale. It has therefore been proposed to synthesize vitamin B2 in a microbiological way. Useful microorganisms for this are, for example, Bacillus subtilis, the Ascomycetes Eremothecium ashbyii, Ashbya gossypu and the yeasts Candida flareri and Saccharomyces cerevisiae. The nutrient media used for this include molasses or vegetable oils as a carbon source, inorganic salts, amino acids, animal or vegetable peptones and proteins as well as vitamin additives. In sterile aerobic submerged processes, yields of more than 10 g of vitamin B2 are obtained within a few days per liter of culture broth. The prerequisites are good ventilation of the culture, careful stirring and setting temperatures below about 30 ° C. After separating the biomass, evaporating and drying the concentrate, a product enriched with vitamin B2 is obtained.

The microbiological production of vitamin B2 is described, for example, in WO-A-92/01060, EP-A-0 405 370 and EP-A-0 531 708.

An overview of the meaning, occurrence, production, biosynthesis and use of vitamin B2 can be found, for example, in Ullmann's Encyclopaedia of Industrial Chemistry, volume A27, pages 521 ff.

The cell wall and the cytoskeleton serve a eukaryotic cell primarily for the maintenance of the external and internal structure. The functions of these components can be compared with those of a tarpaulin and the associated tent poles. However, since living cells do not have a rigid, but because of growth and environmental conditions required, flexible and adaptable cell structure, the structure and composition of external factors, such as Temperature and pH value, but also influenced by internal factors such as the ATP content or the ion concentration of the cell.

The fungal cell wall plays a crucial role during the growth, development or interaction of the fungus with the environment or with other cells. It primarily has a protective function, ie protecting the cell against osmotic, chemical or biological damage. Furthermore, the cell wall is also involved in morphological responses, antigen expression, adhesion and cell-cell interaction. The fungal cell wall is made up of a mixture of different polymers. There are two categories. On the one hand in the so-called structural polymers which are responsible for the rigidity of the structure, and on the other hand in the matrix polymers in which they are embedded and which ensure pressure resistance. For most fungi, chitin, glucans and mannoproteins are the most important cell wall components. Chitin and glucans take on a structural function. The cell wall synthesis takes place via a composition of the individual components in different stages. The individual components must first be synthesized intracellularly or at the plasma / wall boundary layer. After all the polymers have been secreted into the expanding wall, they are initially stored loosely via molecular interactions together before they are firmly connected to each other via covalent bonds.

The cytoskeleton, on the other hand, is a coordinated network of filamentous polymers which are linked to other cellular structures by different molecules. The organization and properties of this network are under precise development-dependent and functional control. The main structural components of the cytoskeleton are the actin filaments (F-actin), microtubules and the intermediate filaments. The cytosol can be compared to a highly organized gel rather than a homogeneous solution, in which the composition can differ significantly in different regions of the cell. The cytoskeleton takes on important tasks in this structuring, but also in cell division and organ transport. In the figurative sense, it takes on the function of railroad tracks on which the most diverse cell components move along with the help of cell motors such as dynein or kinesin.

In contrast to the cell wall structure, the structure of the cytoskeleton is not characterized by the formation of covalent bonds. Since it must have a much greater flexibility, it is characterized by "dynamic instability" as in the case of microtubules. Tubulin subunits are polymerized using GTP. However, since the GTP has the property of decaying to GDP + Pi under cell physiological conditions, the structure of the microtubules is also weakened, so that they must therefore be continuously synthesized in order to then decay again. Proteins associated with microtubules (MAP) enable the cell to achieve stronger or controllable stabilization of the microtubules. Depending on the degree of phosphorylation, MAP's have a high or low affinity and thus a controllable stabilizing effect on the microtubules.

The polymerization of microfilaments from actin and the regulation of the stability of these polymers takes place in the cell analogously to that of tubulin. The polymerization process, on the other hand, is accelerated by ATP. Actin-binding proteins influence the build-up and breakdown of the microfilaments and, as in the case of the profilin, can even prevent the actin from polymerizing.

During development-specific or environmental morphology changes of fungi, for example in buds, fruiting body or pseudo-hyphae development, there are massive restructurings in cell wall synthesis as well as in cytoskeleton development, which are subject to highly precise temporal and spatial regulation. The basic structural framework of the cell is essential for the stability of the cell, the vesicle transport and forms the basic prerequisite for the production of biomass. For a more detailed description of cell wall structure and cytoskeleton structuring, see Wessels, JGH (1990), Role of cell wall architecture in fungal tip growth generation. In: Heath IB (ed) Tip growth in plant and fungal cells. Academic Press, San Diego, pp 1-29; Heath IB and Heath MC (1978), Microtubules and organeile movement in the rustfungus Uromyces phaseoli var. Vignae. Cytobiology 16: 393-411; McConnel SJ, Yaffe MP (1993), Intermediate filament formation by a yeast protein essential for organic inheritance. Science 260: 687-689; Esser K. and Lemke PA (ed) The Mycota - A comprehensive Treatise on fungi as experimental Systems for basic and applied research. Springer-Verlag, Berlin; Voet D. and VoetJ.G. (ed) bio-chemistry. VCH, Weinheim, and the references contained in each of these citations.

The use of genes associated with the synthesis of the cell wall and / or the cytoskeleton for the generation of microorganisms, preferably of the genus Ashbya, especially of Ashbya gossypii strains, with modified cytoskeleton or modified cell wall and e.g. associated modified (higher) resistance to external influences has not yet been described.

The object of the present invention is therefore to provide new targets for influencing the cell wall and cytoskeleton properties in microorganisms of the genus Ashbya, in particular in Ashbya gossypii. In particular, there is the task of improving the stability of the cells in such microorganisms. Another task is the improvement of vitamin B2 production by such microorganisms.

The above object is achieved by providing coding nucleic acid sequences which are up or regulated in Ashbya gossypii during vitamin B2 production (based on results determined using the MPSS analysis method described in more detail in the experimental part), in particular:

a) a, preferably upregulated, nucleic acid sequence which codes for a protein with the function of a cell wall precursor protein.

According to a preferred embodiment of this aspect of the invention, a DNA clone was isolated which codes for a characteristic partial sequence of the nucleic acid sequence according to the invention and which bears the internal name “Oligo 8”. According to a further preferred embodiment, a DNA clone was isolated according to the invention which codes for the full sequence of the nucleic acid according to the invention and which bears the internal name “Oligo 8v”.

A first subject of the present invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 1. Another subject of the invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 4 or a fragment thereof. The polynucleotides can preferably be isolated from a microorganism of the genus Ashbya, in particular A. gossypii. The invention also relates to the complementary polynucleotides; and the sequences derived from these polynucleotides by degenerating the genetic code.

The inserts of "Oligo 8" and "Oligo 8v" have significant homologies with the MIPS tag "Cwp1" from S. cerevisiae. The inserts have a nucleic acid sequence according to SEQ ID NO: 1 and SEQ ID NO: 4, respectively. Those of the complementary strand (Counter strand) to SEQ ID NO: 1 or the amino acid sequence or partial amino acid sequence derived from the coding strand according to SEQ ID NO: 4 has significant sequence homology with the cell wall precursor protein Cwp1 from S. cerevisiae, described by Shimoni H., et al. , in J. Biochem. 118: 302-311 (1995)

b) a, preferably upregulated, nucleic acid sequence which codes for a protein with the function of a serine-threonine kinase.

According to a preferred embodiment of this aspect of the invention, a DNA clone was isolated which codes for a characteristic partial sequence of the nucleic acid sequence according to the invention and which bears the internal name “Oligo 25/39”.

According to a further preferred embodiment, a DNA clone was isolated according to the invention which codes for the full sequence of the nucleic acid according to the invention and which bears the internal name “Oligo 25 / 39v”.

A first subject of the present invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 8. Another subject of the invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 10 or a fragment thereof. The polynucleotides are preferably from a microorganism of the genus Ashbya, in particular A. gossypii. isolated. The invention also relates to the complementary polynucleotides; and the sequences derived from these polynucleotides by degenerating the genetic code. The inserts of "Oligo 25/39" and "Oligo 25 / 39v" have significant homologies with the MIPS tag "ARK1" from S. cerevisiae. The inserts have a nucleic acid sequence according to SEQ ID NO: 8 and SEQ ID NO: 10, respectively. The amino acid sequence derived from the corresponding counter strand to SEQ ID NO: 8 or from the coding strand of SEQ ID NO: 10 has significant sequence homology with a serine-threonine protein kinase from S. cerevisiae.

c) a, preferably upregulated, nucleic acid sequence which codes for a protein with the function of a GTPase-activating protein.

According to a preferred embodiment of this aspect of the invention, a DNA clone was isolated which codes for a characteristic partial sequence of the nucleic acid sequence according to the invention and which bears the internal name “Oligo 46”.

According to a further preferred embodiment, a DNA clone was isolated according to the invention which codes for the full sequence of the nucleic acid according to the invention and which bears the internal name “Oligo 46v”.

A first subject of the present invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 12. Another subject of the invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 15 or a fragment thereof. The polynucleotides can preferably be isolated from a microorganism of the genus Ashbya, in particular A. gossypii. The invention also relates to the complementary polynucleotides; and the sequences derived from these polynucleotides by degeneracy of the genetic code.

The inserts of "Oligo 46" and "Oligo 46v" have significant homologies with the MIPS tag "BUD2 / CLA2" from S. cerevisiae. The inserts have a nucleic acid sequence as shown in SEQ ID NO: 12 and SEQ ID NO: 15, respectively The corresponding counter strand of SEQ ID NO: 12 or of the coding strand derived from SEQ ID NO: 15 has an amino acid sequence or partial amino acid sequence which has significant sequence homology with a GTPase-activating protein from S. cerevisiae, in particular homology to the GTPase-activating protein encoded by BUD2 BUD2 / Rsr1 described by Park H.-O., et al., In Nature 365: 269-274, (1993).

d) a, preferably upregulated, nucleic acid sequence which codes for a protein with the function of resistance to actin overexpression. According to a preferred embodiment of this aspect of the invention, a DNA clone was isolated which codes for a characteristic partial sequence of the nucleic acid sequence according to the invention and which bears the internal name “Oligo 103”.

According to a further preferred embodiment, a DNA clone was isolated according to the invention which codes for the full sequence of the nucleic acid according to the invention and which bears the internal name “Oligo 103v”.

A first subject of the present invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 17. Another subject of the invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 19 or a fragment thereof. The polynucleotides can preferably be isolated from a microorganism of the genus Ashbya, in particular A. gossypii. The invention also relates to the complementary polynucleotides; and the sequences derived from these polynucleotides by degenerating the genetic code.

The inserts of "Oligo 103" and "Oligo 103v" have significant homologies with the MIPS tag "Aor1" from S. cerevisiae. The inserts have a nucleic acid sequence according to SEQ ID NO: 17 and SEQ ID NO: 19. That of the corresponding opposite strand for SEQ ID NO: 17 or the amino acid sequence or partial amino acid sequence derived from the coding strand according to SEQ ID NO: 19 has significant sequence homology with a protein from S. cerevisiae which has resistance to actin overexpression or contributes to this resistance.

e) a, preferably downregulated, nucleic acid sequence which codes for a protein with the function of a protein similar to Nuflp.

According to a preferred embodiment of this aspect of the invention, a DNA clone was isolated which codes for a characteristic partial sequence of the nucleic acid sequence according to the invention and which bears the internal name “Oligo 128”.

According to a further preferred embodiment, a DNA clone was isolated according to the invention which codes for the full sequence of the nucleic acid according to the invention and which bears the internal name “Oligo 128v”. A first subject of the present invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 21. Another subject of the invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 23 or a fragment thereof. The polynucleotides can preferably be isolated from a microorganism of the genus Ashbya, in particular A. gossypii. The invention also relates to the complementary polynucleotides; and the sequences derived from these polynucleotides by degenerating the genetic code.

The inserts of "Oligo 128" and "Oligo 128v" have significant homologies with the MIPS tag "Ykl179c" from S. cererevisiae. The inserts have a nucleic acid sequence according to SEQ ID NO: 21 and SEQ ID NO: 23, respectively coding strand derived amino acid sequence or partial amino acid sequence has significant sequence homology with a protein from S. cerevisiae similar to Nuflp (cf. Wiemann S., et al., Yeast 9: 1343-1348 (1993)).

f) a, preferably upregulated, nucleic acid sequence which is suitable for a protein with the

Function encoded by calponin or a protein homologous to calponin.

According to a preferred embodiment of this aspect of the invention, a DNA clone was isolated which codes for a characteristic partial sequence of the nucleic acid sequence according to the invention and which bears the internal name “Oligo 150”.

According to a further preferred embodiment, a DNA clone was isolated according to the invention which codes for the full sequence of the nucleic acid according to the invention and which bears the internal name “Oligo 150v”.

A first subject of the present invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 26. Another subject of the invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 28 or a fragment thereof. The polynucleotides can preferably be isolated from a microorganism of the genus Ashbya, in particular A. gossypii. The invention also relates to the complementary polynucleotides; and the sequences derived from these polynucleotides by degenerating the genetic code.

The inserts of "Oligo 150" and "Oligo 150v" have significant homologies with the MIPS tag "Scp1" from S. cerevisiae. The inserts have a nucleic acid sequence according to SEQ ID NO:

26 or SEQ ID NO: 28. The amino acid sequences derived in each case from the coding strand zen has significant sequence homology with a calponin or protein from S. cerevisiae homologous to calponin.

g) a, preferably up-regulated, nucleic acid sequence which codes for a protein which is essential for the pseudohyphene development in Candida maitose.

According to a preferred embodiment of this aspect of the invention, a DNA clone was isolated which codes for a characteristic part-sequence of the nucleic acid sequence according to the invention and which bears the internal name “Oligo 177”.

According to a further preferred embodiment, a DNA clone was isolated according to the invention which codes for the full sequence of the nucleic acid according to the invention and which bears the internal name “Oligo 177v”.

A first subject of the present invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 30. Another subject of the invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 34. The polynucleotides are preferably from a microorganism of the genus Ashbya, especially A. gossypii isolable. The invention also relates to the complementary polynucleotides; and the sequences derived from these polynucleotides by degenerating the genetic code.

The inserts of "Oligo 177" and "Oligo 177v" have significant homologies with the MIPS tag "EPD1" from Candida maltosa. The inserts have a nucleic acid sequence according to SEQ ID NO: 30 and SEQ ID NO: 34, respectively. From the corresponding opposite strand of SEQ ID NO: 30 or amino acid sequences which can be derived from the coding strand according to SEQ ID NO: 34 have significant sequence homology with a protein from Candida maltosa, in particular with a protein which is essential for the development of pseudohyphae in C. maltosa (cf. kazawa T., et al., J. Bacteriol., 180 (8), 2079-2086, (1998). Homology to a corresponding protein from S. cerevisiae was also found.

h) a, preferably down-regulated, nucleic acid sequence which codes for a protein with the function of a protein interacting with actin.

According to a preferred embodiment of this aspect of the invention, a DNA clone was isolated which codes for a characteristic partial sequence of the nucleic acid sequence according to the invention and which bears the internal name “Oligo 145”. According to a further preferred embodiment, a DNA clone was isolated according to the invention which codes for the full sequence of the nucleic acid according to the invention and which bears the internal name “Oligo 145v”.

A first subject of the present invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 36. Another subject of the invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 38 or a fragment thereof. The polynucleotides can preferably be isolated from a microorganism of the genus Ashbya, in particular A. gossypii. The invention also relates to the complementary polynucleotides; and the sequences derived from these polynucleotides by degenerating the genetic code.

The inserts of "Oligo145" and "Oligo 145v" have significant homologies with the MIPS tag "Aip2" from S. cerevisiae. The inserts have a nucleic acid sequence as shown in SEQ ID NO: 36 and SEQ ID NO: 38, respectively Strand-derived amino acid sequence or partial amino acid sequence has significant sequence homology with a protein from S. cerevisiae which interacts with actin (cf. Chelstowska A., et al., Yeast 15 (13), 1377-1391 (1999)).

Another object of the invention relates to oligonucleotides which hybridize with one of the above polynucleotides, in particular under stringent conditions.

The invention further relates to polynucleotides which hybridize with one of the oligonucleotides according to the invention and code for a gene product from microorganisms of the genus Ashbya or a functional equivalent of this gene product.

The invention further relates to polypeptides or proteins which are encoded by the polynucleotides described above; and peptide fragments thereof, which have an amino acid sequence, the at least 10 contiguous amino acid residues according to SEQ ID NO: 2, 3, 5, 6, 7, 9, 11, 13, 14, 16, 18, 20, 22, 24, 25, 27 , 29, 31, 32, 33, 35, 37, or SEQ ID NO: 39; and functional equivalents of the polypeptides or proteins according to the invention.

Functional equivalents differ from the products specifically disclosed according to the invention in their amino acid sequence by addition, insertion, substitution, deletion or

Inversion at at least one, for example 1 to 30 or 1 to 20 or 1 to 10, sequence positions without the originally observed and derivable by sequence comparison with other proteins re losing protein function. This means that equivalents can have essentially identical, higher or lower activities compared to the native protein.

Further objects of the invention relate to expression cassettes for the recombinant production of proteins according to the invention, comprising, in operative linkage with at least one regulatory nucleic acid sequence, one of the nucleic acid sequences defined above; as well as recombinant vectors comprising at least one such expression cassette according to the invention.

According to the invention, prokaryotic or eukaryotic hosts are also provided which are transformed with at least one vector of the above type. According to a preferred embodiment, such prokaryotic or eukaryotic hosts are provided in which the functional expression of at least one gene is modulated (e.g. inhibition or overexpression) which codes for a polypeptide according to the invention as defined above; or in which the biological activity of a polypeptide is reduced or increased as defined above. Preferred hosts are selected from Ascomycetes (tubular mushrooms), in particular those of the genus Ashbya and preferably strains of A. gossypii.

Modulation of gene expression in the above sense includes both its inhibition, e.g. by blocking an expression level (in particular transcription or translation) or by deliberately overexpressing a gene (e.g. by modifying regulatory sequences or increasing the number of copies of the coding sequence).

The invention further relates to the use of an expression cassette according to the invention, a vector according to the invention or a host according to the invention for the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof.

Another object of the invention relates to the use of an expression cassette according to the invention, a vector according to the invention or a host according to the invention for the recombinant production of a polypeptide according to the invention as defined above.

According to the invention, a method for the detection or validation of an effector target for the modulation of the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof is also provided. A microorganism which is capable of microbiological production of vitamin B2 and / or precursors and / or derivatives thereof is treated with an effector which has a target selected from a polypeptide according to the invention as defined above or a nucleic acid coding for it. quenz, interacts (such as non-covalently binds to them), validates the influence of the effector on the amount of microbiologically produced vitamin B2 and / or the precursor and / or a derivative thereof; and optionally isolating the target. The validation is preferably carried out by direct comparison with the microbiological vitamin B2 production in the absence of the effector under otherwise identical conditions.

Another object of the invention relates to a method for modulating (in terms of quantity and / or speed) the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof, using a microorganism which is used for the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof is treated with an effector which interacts with a target selected from a polypeptide according to the invention as defined above or a nucleic acid sequence coding therefor.

Preferred examples of the above-mentioned effectors are: a) antibodies or antigen-binding fragments thereof; b) polypeptide ligands which differ from a) and which interact with a polypeptide according to the invention; c) low molecular weight effectors which modulate the biological activity of a polypeptide according to the invention; d) antisense nucleic acid sequences which interact with a nucleic acid sequence according to the invention.

The above-mentioned effectors with specificity for at least one of the inventive targets defined above are also the subject of the invention.

Another object of the invention relates to a method for the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof, wherein a host is cultivated according to the above definition under conditions which favor the production of vitamin B2 and / or precursors and / or derivatives thereof and isolate the desired product (s) from the culture batch. It is preferred that the host is treated with an effector according to the above definition before and / or during cultivation. A preferred host is selected from microorganisms of the genus Ashbya; especially transformed, as described above. A final object of the invention relates to the use of a polynucleotide or polypeptide according to the invention as a target for modulating the production of vitamin B2 and / or precursors and / or derivatives thereof in a microorganism of the genus Ashbya.

Brief Description:

FIG. 1 shows an alignment between an amino acid partial sequence according to the invention (corresponding to the counter strand to positions 1092 to 595 in SEQ ID NO: 1) (upper sequence) and a partial sequence of the MIPS tag “Cwp1” from S. cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with "+".

FIG. 2 shows an alignment between an amino acid partial sequence according to the invention (corresponding to the counter strand to positions 1067 to 84 in SEQ ID NO: 8) (upper sequence) and a partial sequence of the MIPS tag ARK1 from S. cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with "+".

FIG. 3A shows an alignment between an amino acid partial sequence according to the invention (corresponding to the counter strand to positions 475 to 353 in SEQ ID NO: 12) (upper sequence) and a partial sequence of the MIPS tag BUD2 / CLA2 from S. cerevisiae (lower sequence ). FIG. 3B shows an alignment between a partial amino acid sequence according to the invention (corresponding to the counter strand to position 351 to 1 in SEQ ID NO: 12) (upper sequence) and a partial sequence of the MIPS tag BUD2 / CLA2 from S. cerevisiae (lower sequence). Identical sequence positions are given between the two sequences. Similar sequence positions are marked with "+".

FIG. 4 shows an alignment between an amino acid partial sequence according to the invention (corresponding to the counter strand to positions 933 to 157 in SEQ ID NO: 17) (upper sequence) and a partial sequence of the MIPS tag Aor1 from S. cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with "+".

FIG. 5 shows an alignment between a partial amino acid sequence according to the invention (corresponding to the strand to positions 117 to 794 in SEQ ID NO: 21) (upper sequence) and one

Partial sequence of the MIPS tag Ykl179c from S. cerevisiae (lower sequence). Identical sequence Positions are indicated between the two sequences. Similar sequence positions are marked with "+".

FIG. 6 shows an alignment between an amino acid partial sequence according to the invention (corresponding to the strand to positions 438 to 767 in SEQ ID NO: 26) (upper sequence) and a partial sequence of the MIPS tag Scp1 from S. cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with "+".

FIG. 7A shows an alignment between a partial amino acid sequence according to the invention (corresponding to the counter strand to positions 983 to 651 in SEQ ID NO: 30) (upper sequence) and a partial sequence of the MIPS tag EPD1 from C. maltosa (lower sequence). FIG. 7B shows an alignment between an amino acid partial sequence according to the invention (corresponding to the counter strand to positions 661 to 596 in SEQ ID NO: 30) (upper sequence) and a partial sequence of the MIPS tag EPD1 from C. maltosa (lower sequence). FIG. 7C shows an alignment between an amino acid partial sequence according to the invention (corresponding to the counter strand to position 591 to 1 in SEQ ID NO: 30) (upper sequence) and a partial sequence of the MIPS tag EPD1 from C. maltosa (lower sequence). Identical sequence positions are given between the two sequences. Similar sequence positions are marked with "+".

FIG. 8 shows an alignment between an amino acid partial sequence according to the invention (corresponding to the strand to positions 2 to 148 in SEQ ID NO: 36) (upper sequence) and a partial sequence of the MIPS tag Aip2 from S. cerevisiae (lower sequence). Identical sequence positions are indicated between the two sequences. Similar sequence positions are marked with "+".

Detailed description of the invention:

The nucleic acid molecules according to the invention encode proteins or proteins which are referred to here as proteins of the cell wall or cytoskeleton structure (for example with activity relating to cell wall synthesis or cytoskeleton structure) or briefly as “ZC proteins”. These ZC proteins have a function, for example the synthesis or restructuring of the cell wall or cytoskeleton, for example in the case of development-specific or environmental morphological changes in the cell, due to the availability of cloning vectors which can be used in Ashbya gossypii, as disclosed, for example, in Wright and Philipsen (1991) Gene, 109, 99-105., and von Techniques for the genetic manipulation of A. gossypii and the related types of yeast can be used in the nucleic acid molecules according to the invention for the genetic manipulation of these organisms, especially use of A. gossypii to make them better and more efficient as producers of vitamin B2 and / or precursors and / or derivatives thereof. This improved production or efficiency can take place due to a direct effect of the manipulation of a gene according to the invention or due to an indirect effect of such a manipulation.

The present invention is based on the provision of new molecules, referred to here as ZC nucleic acids and ZC proteins, which are involved in the construction of the cell wall and cytoskeleton, in particular in Ashbya gossypii (e.g. in the synthesis or restructuring of the cell wall and cytoskeleton). The activity of the ZC molecules according to the invention in A. gossypii influences the vitamin B2 production by this organism. The activity of the ZC molecules according to the invention is preferably modulated such that the metabolic and / or energy pathways of A. gossypii in which the ZC proteins according to the invention participate are modulated with regard to the yield, production and / or efficiency of vitamin B2 production , which directly or indirectly modulates the yield, production and / or efficiency of vitamin B2 production in A. gossypii.

The nucleic acid sequences provided according to the invention can be isolated, for example, from the genome of an Ashbya gossyp // strain which is freely available from the American Type Culture Collection under the name ATCC 10895.

Improving vitamin B2 production:

There are a number of possible mechanisms by which the yield, production and / or efficiency of the production of vitamin B2 by an A. gossypii strain can be directly influenced by changing the amount and / or activity of a ZC protein according to the invention.

Through a more efficient synthesis of the cell wall and cytoskeleton, the cell can be made more robust against external influences, so that the viability and thus the productivity in the fermenter is increased.

The mutagenesis of one or more ZC proteins according to the invention can also lead to ZC proteins with modified (increased or decreased) activities which indirectly influence the production of the desired product from A. gossypii. For example, the stability of the cells and the vesicle transport in the cells can be adapted to the respective environmental or culture conditions with the help of the ZC proteins and thus the function of essential metabolic processes can be maintained. In addition to the biosynthesis of the The product also includes the structure of the cell walls, transcription, translation, the biosynthesis of compounds that are necessary for the growth and division of cells (e.g. nucleotides, amino acids, vitamins, lipids, etc.) (Lengeieretal. (1999)). By improving the growth and multiplication of these altered cells, it is possible to increase the viability of the cells in cultures on a large scale and also to improve their division rate in such a way that a comparatively larger number of producing cells can survive in the fermenter culture. The yield, production or efficiency of production can be increased at least due to the presence of a larger number of viable cells, each of which produces the desired product.

polypeptides

The invention relates to polypeptides which comprise the above-mentioned amino acid sequences or characteristic partial sequences thereof and / or are encoded by the nucleic acid sequences described herein.

Also included according to the invention are “functional equivalents” of the specifically disclosed new polypeptides.

"Functional equivalents" or analogs of the specifically disclosed polypeptides are, within the scope of the present invention, different polypeptides which furthermore have the desired biological activity (such as substrate specificity).

According to the invention, “functional equivalents” are understood to mean, in particular, mutants which have an amino acid other than the one specifically mentioned in at least one of the sequence positions mentioned above, but nevertheless have one of the biological activities mentioned above. "Functional equivalents" thus encompass the mutants obtainable by one or more amino acid additions, substitutions, deletions and / or inversions, the changes mentioned being able to occur in any sequence position as long as they lead to a mutant with the property profile according to the invention. Functional equivalence is particularly given when the reactivity patterns between mutant and unchanged polypeptide match qualitatively, i.e. for example, the same substrates can be implemented at different speeds.

"Functional equivalents" in the above sense are also precursors of the polypeptides described and functional derivatives and salts of the polypeptides. The term "salts" means both salts of carboxyl groups and acid addition salts of amino nogroups of the protein molecules according to the invention. Salts of carboxyl groups can be prepared in a manner known per se and include inorganic salts, such as, for example, sodium, calcium, ammonium, iron and zinc salts, and salts with organic bases, such as, for example, amines, such as triethanolamine, arginine, lysine , Piperidine and the like. Acid addition salts, such as, for example, salts with mineral acids, such as hydrochloric acid or sulfuric acid, and salts with organic acids, such as acetic acid and oxalic acid, are also a subject of the invention.

"Functional derivatives" of polypeptides according to the invention can also be prepared on functional amino acid side groups or on their N- or C-terminal end using known techniques. Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxyl groups, produced by reaction with acyl groups.

"Functional equivalents" naturally also include polypeptides that are accessible from other organisms, as well as naturally occurring variants. For example, regions of homologous sequence regions can be determined by sequence comparison and equivalent enzymes can be determined based on the specific requirements of the invention.

"Functional equivalents" also include fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention which, for example, have the desired biological function.

“Functional equivalents” are also fusion proteins which contain one of the abovementioned polypeptide sequences or functional equivalents derived therefrom and at least one further, functionally different, heterologous sequence in functional N- or C-terminal linkage (ie without mutual substantial functional impairment of the fusion protein Non-limiting examples of such heterologous sequences are, for example, signal peptides, enzymes, immunoglobulins, surface antigens, receptors or receptor ligands.

"Functional equivalents" encompassed according to the invention are homologs to the specifically hard proteins. These have at least 60%, preferably at least 75%, in particular at least 85%, such as 90%, 95% or 99%, homology to one of the specifically disclosed Sequences calculated according to the algorithm by Pearson and Lipman, Proc. Natl. Acad, Be. (USA) 85 (8), 1988, 2444-2448.

In the case of possible protein glycosylation, equivalents according to the invention include proteins of the type described above in deglycosylated or glycosylated form and also modified forms obtainable by changing the glycosylation pattern.

Homologs of the proteins or polypeptides of the invention can be generated by mutagenesis, e.g. by point mutation or shortening of the protein. The term "homolog" as used here refers to a variant form of the protein which acts as an agonist or antagonist of protein activity.

Homologs of the proteins of the invention can be obtained by screening combinatorial libraries of mutants, e.g. Shortening mutants can be identified. For example, a varied library of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, e.g. by enzymatically ligating a mixture of synthetic oligonucleotides. There are a variety of methods that can be used to generate banks of potential homologs from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automated DNA synthesizer, and the synthetic gene can then be ligated into an appropriate expression vector. The use of a degenerate gene set allows all sequences to be provided in a mixture which encode the desired set of potential protein sequences. Methods for the synthesis of degenerate oligonucleotides are known to the person skilled in the art (eg Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 11: 477).

In addition, banks of fragments of the protein codon can be used to generate a varied population of protein fragments for the screening and subsequent selection of homologues of a protein according to the invention. In one embodiment, a bank of coding sequence fragments can be obtained by treating a double-stranded PCR fragment of a coding sequence with a nuclease under conditions under which nicking occurs only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA Sense / antisense pairs of different nodded products can be removed, single-stranded sections removed from newly formed duplexes by treatment with S1 nuclease and ligating the resulting fragment library into an expression vector. Through this procedure, a Expression bank can be derived, which encodes N-terminal, C-terminal and internal fragments with different sizes of the protein according to the invention.

Several techniques are known in the prior art for screening gene products of combinatorial banks which have been produced by point mutations or truncation, and for screening cDNA banks for gene products with a selected property. These techniques can be adapted to the rapid screening of the gene banks which have been generated by combinatorial mutagenesis of homologues according to the invention. The most commonly used techniques for screening large libraries that are subject to high throughput analysis include cloning the library into replicable expression vectors, transforming the appropriate cells with the resulting vector library, and expressing the combinatorial genes under conditions under which the detection of the desired activity isolation of the vector encoding the gene whose product has been detected is facilitated. Recursive ensemble mutagenesis (REM), a technique that increases the frequency of functional mutants in banks, can be used in combination with the screening tests to identify homologues (Arkin and Yourvan (1992) PNAS 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

The polypeptides according to the invention can be produced recombinantly (cf. the following sections) or can be in native form using conventional biochemical procedures (cf. Cooper, TG, Biochemical Working Methods, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R. , Protein Purification, Springer Verlag, New York, Heidelberg, Berlin) from microorganisms, in particular those of the genus Ashbya, can be isolated.

Nukleinsäureseguenzen:

The invention also relates to nucleic acid sequences (single and double stranded DNA and RNA sequences, such as cDNA and mRNA) coding for one of the above polypeptides and their functional equivalents, which e.g. are accessible using artificial nucleotide analogs.

The invention relates both to isolated nucleic acid molecules which code for polypeptides or proteins or biologically active sections thereof, and to nucleic acid fragments which can be used, for example, for use as hybridization probes or primers for identifying or amplifying coding nucleic acids according to the invention. The nucleic acid molecules according to the invention can also contain untranslated sequences from the 3 'and / or 5' end of the coding gene region.

An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid and, moreover, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free of chemical precursors or other chemicals be when it's chemically synthesized.

A nucleic acid molecule according to the invention can be isolated using standard molecular biological techniques and the sequence information provided according to the invention. For example, cDNA can be isolated from a suitable cDNA library by using one of the specifically disclosed complete sequences or a section thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In addition, a nucleic acid molecule comprising one of the disclosed sequences or a portion thereof can be isolated by polymerase chain reaction using the oligonucleotide primers created based on this sequence. The nucleic acid amplified in this way can be cloned into a suitable vector and characterized by DNA sequence analysis. The oligonucleotides according to the invention which correspond to an SA nucleotide sequence can also be obtained by standard synthesis methods, e.g. with an automatic DNA synthesizer.

The invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a section thereof.

The nucleotide sequences according to the invention enable the generation of probes and primers which can be used for the identification and / or cloning of homologous sequences in other cell types and organisms. Such probes or primers usually comprise a nucleotide sequence region which, under stringent conditions, can be attached to at least about 12, preferably at least about 25, e.g. about 40, 50 or 75 successive nucleotides of a sense strand of a nucleic acid sequence according to the invention or a corresponding antisense strand are hybridized.

Further nucleic acid sequences according to the invention are derived from SEQ ID NO: 1, 4, 8, 10, 12, 15, 17, 19, 21, 23, 26, 28, 30, 34, 36 or SEQ ID NO: 38 and differ from them by addition, substitution, insertion or deletion of one or more nucleotides, but continue to code for polypeptides with the desired property profile.

Also included according to the invention are those nucleic acid sequences which comprise so-called silent mutations or which have been modified in accordance with the codon usage of a specific source or host organism, in comparison to a specifically named sequence, as well as naturally occurring variants, such as e.g. Splice variants or allele variants, thereof. Sequences obtainable by conservative nucleotide substitutions (i.e. the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility) are also a subject of the invention.

The invention also relates to the molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. These genetic polymorphisms can exist between individuals within a population due to natural variation. These natural variations usually cause a variance of 1 to 5% in the nucleotide sequence of a gene.

Furthermore, the invention also encompasses nucleic acid sequences which hybridize with the above-mentioned coding sequences or are complementary thereto. These polynucleotides can be found when screening genomic or cDNA libraries and, if appropriate, can be multiplied therefrom using suitable primers by means of PCR and then isolated, for example, using suitable probes. Another possibility is the transformation of suitable microorganisms with polynucleotides or vectors according to the invention, the multiplication of the microorganisms and thus the polynucleotides and their subsequent isolation. In addition, polynucleotides according to the invention can also be synthesized chemically.

The property of being able to “hybridize” to polynucleotides means the ability of a poly- or oligonucleotide to bind to an almost complementary sequence under stringent conditions, while under these conditions there are no unspecific bindings between non-complementary partners -100%, preferably 90-100%, complementary The property of complementary sequences to be able to specifically bind to one another is made for example in the Northern or Southern blot technique or in the primer binding in PCR or RT PCR is usually used for this purpose, oligonucleotides with a length of 30 base pairs or more, stringent conditions mean, for example in the Northem blot technique, the use of a washing solution which is 50-70 ° C., preferably 60-65 ° C., for example 0.1x SSC buffer with 0.1% SDS (20x SSC: 3M NaCl, 0.3M Na citrate, pH 7.0) for the elution of non-specifically hybridized cDNA probes or oligonucleotides. As mentioned above, only highly complementary nucleic acids remain bound to one another. The setting of stringent conditions is known to the person skilled in the art and is described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. described.

Another aspect of the invention relates to "antisense" nucleic acids. This comprises a nucleotide sequence that is complementary to a coding "sense" nucleic acid. The antisense nucleic acid can be complementary to the entire coding strand or only to a portion thereof In a further embodiment, the antisense nucleic acid molecule is antisense to a non-coding region of the coding strand of a nucleotide sequence. The term "non-coding region" relates to the sequence segments referred to as 5'- and 3'-untranslated regions.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides long. An antisense nucleic acid of the invention can be constructed by chemical synthesis and enzymatic ligation reactions using methods known in the art. An antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides which are designed to increase the biological stability of the molecules or to increase the physical stability of the duplex which is between the antisense and Sense nucleic acid has arisen. For example, phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleosides that can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-ioduracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-

Carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 3-methylguanine Methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, δ'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-nadenine, N6-is6 -5-oxyacetic acid (v), wybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid ( v), 5-Methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. The antisense nucleic acid can also be produced biologically using an expression vector in which a nucleic acid has been subcloned in the antisense direction. The antisense nucleic acid molecules according to the invention are usually administered to a cell or generated in situ so that they hybridize with or bind to the cellular mRNA and / or a coding DNA, so that the expression of the protein, for example by inhibiting transcription and / or translation , is inhibited.

The antisense molecule can be modified to specifically bind to a receptor or to an antigen that is expressed on a selected cell surface, e.g. by linking the antisense nucleic acid molecule to a peptide or an antibody that binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be administered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is under the control of a strong bacterial, viral or eukaryotic promoter are preferred.

In a further embodiment, the antisense nucleic acid molecule according to the invention is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA, the strands running parallel to one another in contrast to conventional alpha units. (Gaultier et al., (1987) Nucleic Acids Res. 15: 6625-6641). The antisense nucleic acid molecule can also be a 2'-0-methyl ribonucleotide (Inoue et al., (1987) Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987) FEBS Lett 215: 327-330).

The invention also relates to ribozymes. These are catalytic RNA molecules with ribonuclease activity that can cleave a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Ribozymes (for example Hammerhead-Ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591)) can thus be used for the catalytic cleavage of transcripts according to the invention, in order to thereby inhibit the translation of the corresponding nucleic acid. A ribozyme with specificity for a coding nucleic acid according to the invention can be formed, for example, on the basis of a cDNA specifically disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed where the nucleotide sequence of the active site is complementary to the nucleotide sequence that is to be cleaved in a coding mRNA according to the invention. (See, for example, US-A-4 987 071 and US-A-5 116742). Alternatively, mRNA can be used to select a catalytic RNA with specific ribonuclease activity from a pool of RNA molecules (see, for example, Bartel, D., and Szostak, JW (1993) Science 261: 1411-1418). The gene expression of sequences according to the invention can alternatively be inhibited by directing nucleotide sequences which are complementary to the regulatory region of a nucleotide sequence according to the invention (for example to a promoter and / or enhancer of a coding sequence) in such a way that triple helix structures are formed which transcribe of the corresponding gene in target cells (Helene, C. (1991) Anticancer Drug Res. 6 (6) 569-584; Helene, C. et al., (1992) Ann. NY Acad. Sci. 660: 27- 36; and Mower. LJ (1992) Bioassays 14 (12): 807-815).

Expression constructs and vectors:

The invention also relates to expression constructs containing, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a polypeptide according to the invention; and vectors comprising at least one of these expression constructs. Such constructs according to the invention preferably comprise a promoter 5'-upstream of the respective coding sequence and 3'-downstream a terminator sequence and, if appropriate, further customary regulatory elements, in each case operatively linked to the coding sequence. An “operative linkage” is understood to mean the sequential arrangement of promoter, coding sequence, terminator and optionally further regulatory elements in such a way that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended. Examples of sequences which can be linked operatively are Targeting sequences and enhancers, polyadenylation signals and the like. Further regulatory elements include selectable markers, amplification signals, origins of replication and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego , CA (1990).

In addition to the artificial regulatory sequences, the natural regulatory sequence can still be present before the actual structural gene. This natural regulation can possibly be switched off by genetic modification and the expression of the genes increased or decreased. However, the gene construct can also have a simpler structure, ie no additional regulation signals are inserted in front of the structural gene and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation no longer takes place and gene expression is increased or decreased. The nucleic acid sequences can be contained in one or more copies in the gene construct. Examples of useful promoters are: cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5, T3, gal, trc -, ara, SP6, λ-PR or in the λ-PL promoter, which are advantageously used in gram-negative bacteria; as well as the gram-positive promoters amy and SP02, the yeast promoters ADC1, MFα, AC, P-60, CYC1, GAPDH or the plant promoters CaMV / 35S, SSU, OCS, Iib4, usp, STLS1, B33, not or the ubiquitin or phaseolin promoter. The use of inducible promoters, such as, for example, light-inducible and in particular temperature-inducible promoters, such as the P _r P _r promoter, is particularly preferred. In principle, all natural promoters with their regulatory sequences can be used. In addition, synthetic promoters can also be used advantageously.

The regulatory sequences mentioned are intended to enable the targeted expression of the nucleic acid sequences. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.

The regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase or decrease it. Thus, the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers". In addition, an increase in translation is also possible, for example, by improving the stability of the mRNA.

An expression cassette is produced by fusing a suitable promoter with a suitable nucleotide sequence according to the invention and a terminator or polyadenylation signal. Common recombination and cloning techniques, such as those described in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which enables optimal expression of the genes in the host. Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985). In addition to plasmids, vectors also include all other vectors known to the person skilled in the art, such as phages, viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA to understand. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally.

The following can be mentioned as examples of suitable expression vectors:

Common fusion expression vectors such as pGEX (Pharmacia Biotech Ine; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT 5 (Pharmacia, Piscataway, NJ) which glutathione-S-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.

Non-fusion protein expression vectors such as pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California ( 1990) 60-89).

Yeast expression vector for expression in the yeast S. cerevisiae, such as pYepSed (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods of constructing vectors suitable for use in other fungi, such as filamentous fungi, include those described in detail in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector developmentforfilamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy et al., Eds., Pp. 1-28, Cambridge University Press: Cambridge.

Baulovirus vectors available for expression of proteins in cultured insect cells (e.g. Sf9 cells) include the pAc series (Smith et al., (1983) Mol. Cell BioL 3: 2156-2165) and the pVL- Series (Lucklow and Summers (1989) Virology 170: 31-39).

Plant expression vectors, such as those described in detail in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with seleetable markers located proximal to the left border" , Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 8711-8721.

Mammalian expression vectors such as pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). Further suitable expression systems for prokaryotic and eukaryotic cells are in chapters 16 and 17 of Sambrook, J., Fritsch, EF and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY, 1989.

Recombinant microorganisms:

With the aid of the vectors according to the invention, recombinant microorganisms can be produced which, for example, have been transformed with at least one vector according to the invention and can be used to produce the polypeptides according to the invention. The recombinant constructs according to the invention described above are advantageously introduced and expressed in a suitable host system. Common cloning and transfection methods known to the person skilled in the art, such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, are preferably used to bring the nucleic acids mentioned into expression in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

According to the invention, homologously recombined microorganisms can also be produced. For this purpose, a vector is produced which contains at least a section of a gene or a coding sequence according to the invention, in which at least one amino acid deletion, addition or substitution has optionally been introduced in order to change the sequence according to the invention, e.g. functionally disrupt ("Knockouf 'vector). The introduced sequence can also be, for example, a homolog from a related microorganism or derived from a mammalian, yeast or insect source. The vector used for homologous recombination can alternatively be designed such that the endogenous gene is mutated or otherwise altered in homologous recombination, but still encodes the functional protein (for example, the upstream regulatory region can be altered in such a way that the expression of the endogenous protein is thereby altered). The altered section of the ZC gene is in the homologous recombination vector The construction of suitable vectors for homologous recombination is described, for example, in Thomas, KR and Capecchi, MR (1987) Cell 51: 503.

In principle, all organisms which allow expression of the nucleic acids according to the invention, their allele variants, their functional equivalents or derivatives are suitable as host organisms. Among host organisms are, for example, bacteria, fungi, yeasts, vegetable or to understand animal cells. Preferred organisms are bacteria, such as those of the genera Escherichia, such as. B. Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus, higher eukaryotic cells from animals or plants, for example Sf9 or CHO cells. Preferred organisms are selected from the genus Ashbya, in particular from A. gossyp // ^' strains.

Successfully transformed organisms can be selected using marker genes which are also contained in the vector or in the expression cassette. Examples of such marker genes are genes for antibiotic resistance and for enzymes which catalyze a coloring reaction which stains the transformed cell. These can then be selected using automatic cell sorting. Microorganisms successfully transformed with a vector and carrying an appropriate antibiotic resistance gene (e.g. G418 or hygromycin) can be selected using appropriate antibiotic-containing media or nutrient media. Marker proteins that are presented on the cell surface can be used for selection by means of affinity chromatography.

The combination of the host organisms and the vectors which match the organisms, such as plasmids, viruses or phages, such as, for example, plasmids with the RNA polymerase / promoter system, the phages λ or μ or other temperate phages or transposons and / or further advantageous regulatory ones Sequences form an expression system. For example, the term “expression system” means the combination of mammalian cells, such as CHO cells, and vectors, such as pcDNA3neo vector, which are suitable for mammalian cells.

If desired, the gene product can also be expressed in transgenic organisms such as transgenic animals, such as in particular mice, sheep or transgenic plants.

Recombinant production of the polypeptides:

The invention furthermore relates to processes for the recombinant production of a polypeptide according to the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultivated, where appropriate the expression of the polypeptides is induced and these are isolated from the culture. The polypeptides can thus also be produced on an industrial scale, if this is desired.

The recombinant microorganism can be cultivated and fermented by known methods. Bacteria can, for example, in TB or LB medium and at a temperature of 20 to 40 ° C and a pH of 6 to 9 can be increased. Suitable cultivation conditions are described in detail, for example, in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).

If the polypeptides are not secreted into the culture medium, the cells are then disrupted and the product is obtained from the lysate by known protein isolation methods. The cells can optionally be operated by high-frequency ultrasound, by high pressure, e.g. in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by a combination of several of the processes listed.

Purification of the polypeptides can be achieved with known chromatographic methods, such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and with other conventional methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, T.G., Biochemical Working Methods, Walter de Gruyter Verlag, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

To isolate the recombinant protein, it is particularly advantageous to use vector systems or oligonucleotides which extend the cDNA by certain nucleotide sequences and thus code for modified polypeptides or fusion proteins which e.g. serve easier cleaning. Such suitable modifications are, for example, so-called "tags" functioning as anchors, such as the modification known as hexa-histidine anchors, or epitopes that can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press ). These anchors can be used to attach the proteins to a solid support, e.g. a polymer matrix, which can be filled, for example, in a chromatography column, or can be used on a microtiter plate or on another support.

At the same time, these anchors can also be used to recognize the proteins. To recognize the proteins, customary markers, such as fluorescent dyes, enzyme markers which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors, can be used to derivatize the proteins. The invention also relates to a method for the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof.

If the reaction is carried out with a recombinant microorganism, the microorganisms are preferably first cultivated in the presence of oxygen and in a complex medium, such as e.g. at a cultivation temperature of about 20 ° C or more, and a pH of about 6 to 9 until a sufficient cell density is reached. To better control the reaction, the use of an inducible promoter is preferred. The cultivation is continued for 12 hours to 3 days after the induction of vitamin B2 production in the presence of oxygen.

The following non-limiting examples describe specific embodiments of the invention.

General experimental information

a) General cloning procedures

The cloning steps performed in the present invention, such as e.g. Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA were carried out as in Sambrook et al. (1989) op. described.

b) Polymerase chain reaction (PCR)

PCR was carried out according to the standard protocol with the following standard approach:

8 μl dNTP mix (200μM), 10 μl Taq polymerase buffer (10 ×) without MgCI ₂ , 8 μl MgCI ₂ (25mM), 1 μl primer (0.1 μM) each, Iμl DNA to be amplified, 2.5 U Taq polymerase (MBI Fermentas, Vilnius, Lithuania), ad 100 μl demineralized water.

c) Cultivation of E. coli

The cultivation of recombinant E. coli strains DH5α was carried out in LB-Amp medium (trypton 10.0 g, NaCl 5.0 g, yeast extract 5.0 g, ampicillin 100 g / ml H ₂ O ad 1000 ml) at 37 ° C cultured. For this purpose, one colony was transferred from an agar plate into 5 ml LB-Amp using an inoculation loop. After culturing for about 18 hours at a shaking frequency of 220 rpm, 400 ml of medium were inoculated with 4 ml of culture in a 2 l flask. P450 expression was induced in E. coli after an OD578 value between 0.8 and 1.0 was reached by inducing heat shock at 42 ° C. for three to four hours.

d) purification of the desired product from the culture

The desired product can be obtained from the microorganism or from the culture supernatant by various methods known in the art. If the desired product is not secreted by the cells, the cells can be harvested from the culture by slow centrifugation, the cells can be lysed by standard techniques such as mechanical force or ultrasound treatment.

The cell debris is removed by centrifugation and the supernatant fraction containing the soluble proteins is obtained for further purification of the desired compound. If the product is secreted from the cells, the cells are removed from the culture by slow centrifugation and the supernatant fraction is retained for further purification.

The supernatant fraction from both purification processes is subjected to chromatography with a suitable resin, the desired molecule either being retained on the chromatography resin or passing through it with higher selectivity than the impurities. These chromatography steps can be repeated if necessary using the same or different chromatography resins. The person skilled in the art is skilled in the selection of the suitable chromatography resins and their most effective application for a particular molecule to be purified. The purified product can be concentrated by filtration or ultrafiltration and kept at a temperature at which the stability of the product is maximum.

Many cleaning methods are known in the prior art. These cleaning techniques are e.g. described in Bailey, J.E. & Ollis, D.F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined by prior art techniques. These include high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, NIRS, enzyme test or microbiological tests. These analysis methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

e) General description of the MPSS method, clone identification and homology search

MPSS technology (massive parallel signature sequencing, as described by Brenner et al, Nat. Biotechnol. (2000) 18, 630-634; to which express reference is made) was applied to the filamentous mushroom Ashbya gossypii that produces vitamin B2. With the help of this technology it is possible to obtain quantitative statements about the expression strength of a large number of genes in a eukaryotic organism with high accuracy. The mRNA of the organism is isolated at a specific point in time X, transcribed into cDNA using the enzyme reverse transcriptase and then cloned into special vectors which have a specific tag sequence. The number of vectors with different tag sequences is chosen so high (about 1000 times higher) that, statistically speaking, each DNA molecule is cloned into a vector that is unique due to its tag sequence.

Then the vector inserts are cut out together with the tag. The DNA molecules thus obtained are then incubated with microspheres that have the molecular counterparts of the tags mentioned. After incubation, it can be assumed that each microsphere is loaded with only one type of DNA molecule via the specific tags or counterparts. The beads are transferred to a special flow cell and fixed there, so that it is possible to carry out a mass sequencing of all beads using an adapted sequencing method based on fluorescent dyes and using a digital color camera. With this method, a numerically high evaluation is possible, but is limited by a reading range of approximately 16 to 20 base pairs. However, the sequence length is sufficient to allow a clear assignment between sequence and gene in most organisms (20 bp have a sequence frequency of -1x10 ¹² , the human genome has "only" a size of -3x10 ⁹ bp in comparison).

The data obtained in this way are evaluated by counting the number of such sequences and comparing their frequencies with one another. Frequently occurring sequences reflect a high level of expression, occasionally occurring sequences reflect a low level of expression. If the mRNA isolation took place at two different times (X and Y), it is possible to set up a temporal expression pattern of individual genes. Example 1:

Isolation of Ashbya gossypii mRNA

Ashbya gossypii was cultivated in a manner known per se (nutrient medium: 27.5 g / l yeast extract; 0.5 g / l magnesium sulfate; 50 ml / l soybean oil; pH 7). Ashbya gossypii mycelium samples are taken at different times during the fermentation (24h, 48h and 72h) and the corresponding RNA or mRNA is prepared according to the protocol of Sambrook et al. (1989) isolated from it.

Example 2:

Application of the MPSS

A. gossypii mRNA isolated is then subjected to MPSS analysis as explained above.

The determined data sets are subjected to a statistical evaluation and classified according to the significance of the expression differences. Both the increase and decrease in the level of expression were examined. The expression change is classified into a) monotonous change, b) change after 24h, and c) change after 48h.

The 20bp sequences, which represent an expression change and are determined by MPSS analysis, are then used as probes and hybridized against an Ashbya gossypii gene library with an average insert size of approximately 1 kb. The hydriding temperature was in the range from about 30 to 57 ° C.

Example 3:

Creation of a genomic gene bank from Ashbya gossypii

To create a genomic DNA bank, chromosomal DNA is first isolated using the method of Wright and Philippsen (Gene (1991) 109: 99-105) and Mohr (1995, PhD thesis, Biozentrum University Basel, Switzerland).

The DNA is partially digested with Sau3A. For this purpose, 6μg genomic DNA is subjected to Sau3A digestion with different amounts of enzyme (0.1 to 1 U). The fragments are fractionated in a sucrose density gradient. The 1 kb region is isolated and subjected to QiaEx extraction. The largest fragments are ligated with the Ba HI cut vector pRS416 (Sikorski and Hieter, Genetics (1988) 122; 19-27) (90 ng BamHI cut, dephosphorylated vector; 198ng insert DNA; 5ml water; 2 ul 10x ligation buffer; 1 U ligase). With this ligation approach £ co // laboratory strain XL-1 blue is transformed and the resulting clones are used to identify the insert.

Example 4:

Creation of an orderly gene bank (CHIP technology)

About 25,000 colonies from the Ashbya gossypii gene bank (this corresponds to approximately 3 times the genome coverage) were transferred to a nylon membrane in an orderly manner and then transferred using the colony hybridization method as described in Sambrook et al. (1989). Oligonucleotides were synthesized from the ²⁰ bp sequences determined by MPSS analysis and radioactively labeled using ³² P. 10 labeled oligonucleotides with a similar melting point are combined and hybridized together against the nylon membranes. After hybridization and washing steps, positive clones are identified by autoradiography and analyzed directly using PCR sequencing.

In this way, a clone was identified which bears an insert with the internal name "Oligo 8" and which has significant homologies with the MIPS tag "Cwp1" from S. cerevisiae. The insert has a nucleic acid sequence as shown in SEQ ID NO: 1.

In this way, a clone was identified which bears an insert with the internal name "Oligo 25/39" and which has significant homologies with the MIPS tag "ARK1" from S. cerevisiae. The insert has a nucleic acid sequence as shown in SEQ ID NO: 8.

In this way, a clone was identified which bears an insert with the internal name "Oligo 46" and which has significant homologies with the MIPS tag "BUD2 / CLA2" from S. cerevisiae. The insert has a nucleic acid sequence as shown in SEQ ID NO: 12.

In this way, a clone was identified which bears an insert with the internal name “Oligo 103” and has significant homologies with the MIPS tag “Aor1” from S. cerevisiae. The insert has a nucleic acid sequence as shown in SEQ ID NO: 17.

In this way, a clone was also identified which bears an insert with the internal name "Oligo 128" and which has significant homologies with the MIPS tag "Ykl179c" from S. cerevisiae. The insert has a nucleic acid sequence as shown in SEQ ID NO: 21. In this way, a clone was identified which bears an insert with the internal name "Oligo 150" and which has significant homologies with the MIPS tag "Scp1" from S. cerevisiae. The insert has a nucleic acid sequence as shown in SEQ ID NO: 26.

In this way, a clone was identified which bears an insert with the internal name “Oligo 177” and has significant homologies with the MIPS tag “EPD1” from C. maltosa. The insert has a nucleic acid sequence as shown in SEQ ID NO: 30.

In this way, a clone was identified which bears an insert with the internal name "Oligo 145" and which has significant homologies with the MIPS tag "Aip2" from S. cerevisiae. The insert has a nucleic acid sequence as shown in SEQ ID NO: 36.

Example 5:

Evaluation of the sequence data using a BLASTX search

An evaluation of the nucleic acid sequences obtained, i.e. their functional assignment to a functional amino acid sequence was carried out using a BLASTX search in sequence databases. Almost all of the amino acid sequence homologies found concerned Saccharomyces cerevisiae (baker's yeast). Since this organism has already been completely sequenced, more detailed information regarding these genes can be found at: http://www.mips.gsf.de/proi/yeast/search/code search.htm.

The following homologies with an amino acid fragment from S. cerevisiae were determined. The corresponding alignments are shown in the attached FIGS. 1 to 8.

a) The amino acid sequence derived from the coding strand to SEQ ID NO: 1 has significant sequence homology with a cell wall precursor protein from S. cerevisiae. A partial amino acid sequence derived therefrom (corresponding to nucleotides 1092 to 595 from SEQ ID NO: 1) with a partial sequence of the S. cerevisiae protein is shown in FIG. 1. SEQ ID NO: 2 and SEQ ID NO: 3 each show an N-terminally extended amino acid partial sequence.

The A. gossypii nucleic acid sequence determined could thus be assigned the function of a cell wall precursor protein.

b) The amino acid sequence derived from the corresponding counter-strand to SEQ ID NO: 8 has significant sequence homology with a serine-threonine kinase from S. cerevisiae. A partial amino acid sequence derived from it (corresponding to the nucleotides 1067 to 84 from SEQ ID NO: 8) with a partial sequence of the S. cerevisiae enzyme is shown in FIG. 2. SEQ ID NO: 9 shows an N-terminally extended amino acid partial sequence.

The A. gossypii nucleic acid sequence determined could thus be assigned the function of a serine threonine kinase.

c) The amino acid sequence derived from the counter strand to SEQ ID NO: 12 has significant sequence homology with a GTPase-activating protein from S. cerevisiae. A partial amino acid sequence derived therefrom (corresponding to nucleotides 475 to 353 from SEQ ID NO: 12) with a partial sequence of the S. cerevisiae protein is shown in FIG. 3A. Another amino acid part-sequence derived therefrom (corresponding to nucleotides 351 to 1 from SEQ ID NO: 12) with a part-sequence of the S. cerevisiae protein is shown in FIG. 3B. SEQ ID NO: 13 and SEQ ID NO: 14 each show an N-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence determined could thus be assigned the function of a GTPase-activating protein.

d) The amino acid sequence derived from the corresponding counter strand to SEQ ID NO: 17 has significant sequence homology with a protein from S. cerevisiae, which is associated with resistance to actin overexpression. An amino acid partial sequence derived therefrom (corresponding to nucleotides 933 to 157 from SEQ ID NO: 17) with a partial sequence of the S. cerevisiae protein is shown in FIG. 4. SEQ ID NO: 18 shows an N-terminally extended amino acid partial sequence.

The A. gossypii nucleic acid sequence determined could thus be assigned to the function of a protein which is resistant to actin overexpression.

e) The amino acid sequence derived from the coding strand to SEQ ID NO: 21 has significant sequence complicity with a protein from S. cerevisiae similar to Nuflp. An amino acid partial sequence derived therefrom (corresponding to nucleotides 117 to 794 from SEQ ID NO: 21) with a partial sequence of the S. cerevisiae protein is shown in FIG. 5. SEQ ID NO: 22 shows an N-terminally extended amino acid partial sequence.

The A. gossypii nucleic acid sequence determined could thus be assigned the function of a protein similar to Nuf 1 p. f) The amino acid sequence derived from the coding strand to SEQ ID NO: 26 has significant sequence homology with a protein from S. cerevisiae homologous to calponin. An amino acid partial sequence derived therefrom (corresponding to nucleotides 438 to 767 from SEQ ID NO: 26) with a partial sequence of the S. cerevisiae protein is shown in FIG. 6. SEQ ID NO: 27 shows an N-terminally extended amino acid partial sequence.

The A. gossypii nucleic acid sequence determined could thus be assigned the function of a protein homologous to calponin.

g) The amino acid sequence derived from the corresponding counter-strand to SEQ ID NO: 30 has significant sequence homology with a protein from C. maltosa, which is essential for pseudohyphene development in maltosa. An amino acid partial sequence derived therefrom (corresponding to nucleotides 983 to 651 from SEQ ID NO: 30) with a partial sequence of the C. maltosa protein is shown in FIG. 7A. A further partial amino acid sequence derived therefrom (corresponding to nucleotides 661 to 596 from SEQ ID NO: 30) with a partial sequence of the C. maltosa protein is shown in FIG. 7B. A third amino acid partial sequence derived therefrom (corresponding to nucleotides 591 to 1 from SEQ ID NO: 30) with a partial sequence of the C. maltosa protein is shown in FIG. 7C. SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33 each show an N-terminally extended amino acid partial sequence

The A. gossypii nucleic acid sequence determined could thus be assigned to the function of a protein which is essential for the development of pseudohyphae in C. maltosa.

h) The amino acid sequence derived from the coding strand to SEQ ID NO: 36 has significant sequence homology with a protein from S. cerevisiae, which interacts with actin. An amino acid partial sequence derived therefrom (corresponding to nucleotides 2 to 148 from SEQ ID NO: 36) with a partial sequence of the S. cerevisiae protein is shown in FIG. 8. SEQ ID NO: 37 shows an N-terminally extended amino acid partial sequence.

The A. gossypii nucleic acid sequence determined could thus be assigned to the function of a protein which interacts with actin.

Example 6: Isolation of full-length DNA

a) Construction of an A. gossyp / 7 gene bank A. gossypii high molecular weight cellular DNA was prepared from a 2 day old 100 ml culture grown in a liquid MA2 medium (10 g glucose, 10 g peptone, 1 g yeast extract, 0.3 g Myo-Inositad 1000 ml). The mycelium was filtered off, twice with H ₂ 0 dest. washed, suspended in 10 ml of 1M sorbitol, 20 mM EDTA, containing 20 mg of zymolyase-20T, and incubated at 27 ° C. for 30 to 60 min with gentle shaking. The protoplast suspension was adjusted to 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 100 mM EDTA and 0.5% sodium dodecyl sulfate (SDS) and incubated at 65 ° C. for 20 min. After two extractions with phenol-chloroform (1: 1 vol / vol), the DNA was precipitated with isopropanol, suspended in TE buffer, treated with RNase, precipitated again with isopropanol and resuspended in TE.

An A. gossyp // cosmid library was made by binding genomic DNA selected in size, partially digested with Sau3A, to the dephosphorylated arms of the cosmid vector Super-Cos1 (Stratagene). The Super Cos1 vector was opened between the two cos sites by digestion with Xbal and dephosphorylation with alkaline calf intestinal phosphatase (Boehringer), followed by opening the cloning site with BamHI. The ligations were carried out overnight at 15 ° C. in 20 μl, containing 2.5 μg partially digested chromosomal DNA, 1 μg Super-Cos1 vector arms, 40 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 1mM dithiothreitol, 0 , 5 mM ATP and 2 Weiss units T4 DNA ligase (Boehringer). The ligation products were packaged in vitro using the extracts and protocol from Stratagene (Gigapack II Packing Extract). The packaged material was used to infect E. coli NM554 {recA13, araD139, Δ (ara, leu) 7696, Δ (lac) 17A, galil, galK, hsrR, rps (stt ^r ), mcrA, mcrB) and on ampicillin (50 μg / ml) containing LB plates distributed. Transformants were obtained which contained an A. gossyρ / 7 insert with an average length of 30-45 kb.

b) Storage and screening of the Cosmid gene bank

A total of 4 × 10 ⁴ fresh individual colonies were individually in wells of 96-well microtiter plates (Falcon, No. 3072) in 100 μl LB medium, supplemented with the freezing medium (36 mM K ₂ HPO ₄ / 13.2 mM KH ₂ PO ₄ , 1.7 mM sodium citrate, 0.4 mM MgSO ₄ , 6.8 mM (NH ₄ ) ₂ SO ₄ , 4.4% (wt / vol) glycerol) and ampicillin (50 μg / ml), inoculated, overnight Let grow at 37 ° C with shaking and freeze at -70 ° C. The plates were quickly thawed and then duplicated in fresh medium using a 96 series replicator which had been sterilized in an ethanol bath followed by evaporation of the ethanol on a hot plate. Before freezing and after thawing (before any other measures), the plates were briefly shaken in a microtiter shaker (Infors) to ensure a homogeneous cell suspension. By means of a robot system (bio-robotics), with the low Amounts of liquid from 96 wells of a microtiter plate can be transferred to nylon membrane (GeneScreen Plus, New England Nuclear), individual clones were placed on nylon membranes. After the transfer of the culture from the 96-well microtiter plates (1920 clones), the membranes were placed on the surface of LB agar with ampicillin (50 μg / ml) in 22 × 22 cm culture dishes (Nunc) and overnight at 37 ° C. incubated. Before cell confluence was reached, the membranes were processed as described by Herrmann, BG, Barlow, DP and Lehrach, H. (1987) in Cell 48, pp. 813-825, with an additional treatment after the first denaturation step being a 5- minutes of steaming the filters on a pad soaked in denaturing solution is added over a boiling water bath.

With the help of the random hexamer primer method (Feinberg, AP and Vogelstein, B. (1983), Anal. Biochem. 132, pp. 6-13) double-stranded probes were obtained by taking up [alpha- ³² P] dCTP with high specific activity. The membranes were prehybridized and 6 to 12 h at 42 ° C in 50% (vol / vol) formamide, 600 mM sodium phosphate, pH 7.2, 1 mM EDTA, 10% dextran sulfate, 1% SDS, and 10x Denhardt's solution, containing salmon sperm DNA (50 ug / ml) hybridized with ³² P-labeled probes (0.5-1 x 10 ⁶ cpm / ml). Typically, washing steps were carried out for about 1 hour at 55 to 65 ° C. in 13 to 30 mM NaCl, 1.5 to 3 mM sodium citrate, pH 6.3, 0.1% SDS and the filters were 12 to 24 hours at -70 ° C autoradiographed with Kodak amplifier plates. So far, individual membranes have been successfully reused more than 20 times. Between the autoradiographs, the filters were stripped by incubation at 95 ° C for 2 x 20 min in 2 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 0.1% SDS.

c) recovery of positive colonies from the stored gene bank

Frozen bacterial cultures in microtiter wells were scraped using sterile disposable lancets and the material was spread on LB agar petri dishes containing ampicillin (50 μg / ml). Individual colonies were then used to inoculate liquid cultures for the preparation of DNA using an alkali lysis method (Bimboim, H.C. and Doly, J. (1979), Nucleic Acids Res. 7, pp. 1513-1523).

d) Full-length DNA

In the manner described above, clones could be identified which carried an insert with the corresponding full sequence. These clones have the internal names:

"Oligo 8v". The insert comprising the full sequence has a nucleic acid sequence as shown in SEQ ID NO: 4. The protein encoded thereby preferably comprises at least one of the amino Acid sequences according to SEQ ID NO: 5, 6 and 7.

"Oligo 25 / 39v". The insert comprising the full sequence has a nucleic acid sequence as shown in SEQ ID NO: 10.

"Oligo 46v". The insert comprising the full sequence has a nucleic acid sequence as shown in SEQ ID NO: 15.

"Oligo 103v". The insert comprising the full sequence has a nucleic acid sequence as shown in SEQ ID NO: 19.

"Oligo 128v". The insert comprising the full sequence has a nucleic acid sequence as shown in SEQ ID NO: 23. The protein encoded therein preferably comprises at least one of the amino acid sequences as shown in SEQ ID NO: 24 and 25.

"Oligo 150v". The insert comprising the full sequence has a nucleic acid sequence as shown in SEQ ID NO: 28.

"Oligo 177v". The insert comprising the full sequence has a nucleic acid sequence as shown in SEQ ID NO: 34.

"Oligo 145v". The insert comprising the full sequence has a nucleic acid sequence as shown in SEQ ID NO: 38.

Table 1: Sequence overview

Claims

claims

1. Polynucleotide which can be isolated from Ashbya gossypii and which codes for a protein which is associated with the cell wall and / or cytoskeleton structure.

2. Polynucleotide according to claim 1, which is associated with the cell wall and / or cytoskeleton structure and has a structural and / or functional property given in Table 1.

3. Polynucleotide according to claim 1 or 2, comprising a nucleic acid sequence according to

SEQ ID NO: 1, 8, 12, 17, 21, 26, 30 or 36, which can preferably be isolated from Ashbya gossypii; the complementary polynucleotide; and the sequences derived from these polynucleotides by degenerating the genetic code.

4. Polynucleotide according to claim 3, which has a nucleic acid sequence according to SEQ ID

NO: 4, 10, 15, 19, 23, 28, 34 or 38 or a fragment thereof.

5. oligonucleotide which hybridizes with a polynucleotide according to one of the preceding claims, in particular under stringent conditions.

Polynucleotide which hybridizes with an oligonucleotide according to claim 5, in particular under stringent conditions, and codes for a gene product from microorganisms of the genus Ashbya or a functional equivalent of this gene product.

7. A polypeptide encoded by a polynucleotide comprising a nucleic acid sequence according to any one of claims 1 to 4 or a fragment thereof, or encoded by a polynucleotide according to claim 6; or which has an amino acid sequence which has at least 10 contiguous amino acid residues according to SEQ ID NO: 2, 3, 5, 6, 7, 9, 11, 13, 14, 16, 18, 20, 22, 24,

25, 27, 29, 31, 32, 33, 35, 37, or SEQ ID NO: 39; as well as functional equivalents thereof, in particular those which have one of the properties defined in claim 2.

8. Expression cassette comprising, in operative linkage with at least one regulatory nucleic acid sequence, a nucleic acid sequence according to one of claims 1 to 6.

9. A recombinant vector comprising at least one expression cassette according to claim 8.

10. Prokaryotic or eukaryotic host transformed with at least one vector according to claim 9.

11. Prokaryotic or eukaryotic host in which the functional expression of at least one gene is modulated which codes for a polypeptide according to claim 7; or in which the biological activity of a polypeptide according to claim 7 is decreased or increased.

12. The host of claim 10 or 11 from the genus Ashbya.

13. Use of an expression cassette according to claim 8, a vector according to claim 9 or a host according to one of claims 10 to 12 for the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof.

14. Use of an expression cassette according to claim 8, a vector according to claim 9 or a host according to one of claims 10 to 12 for the recombinant

Preparation of a polypeptide according to claim 7.

15. A method for detecting an effector target for the modulation of the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof, using a microorganism which is responsible for the microbiological production of

Vitamin B2 and / or precursors and / or derivatives thereof is capable of being treated with an effector which interacts, in particular binds, with a target selected from a polypeptide according to claim 7 or a nucleic acid sequence coding therefor, the influence of the effector on the amount of validated microbiologically produced vitamin B2, and / or the precursor and / or a derivative thereof; and optionally isolating the target.

16. A method for modulating the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof, using a microorganism which is responsible for the microbiological production of vitamin B2 and / or precursors and / or

Derivatives thereof is capable of being treated with an effector which interacts with a target selected from a polypeptide according to claim 7 or a nucleic acid sequence coding therefor.

17. Effector for a target, selected from a polypeptide according to claim 7 or a nucleic acid sequence coding therefor, the effector being selected from: a) antibodies or antigen-binding fragments thereof; b) polypeptide ligands which differ from a) and which interact with the polypeptide according to claim 7; c) low molecular weight effectors which modulate the biological activity of a polypeptide according to claim 7; d) antisense nucleic acid sequences.

18. A method for the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof, wherein a host is cultivated according to one of claims 10 to 12 under the conditions favorable to the production of vitamin B2 and / or precursors and / or derivatives thereof and that The desired product (s) isolated from the culture batch.

19. The method according to claim 18, wherein before and / or during the cultivation of the host, the latter is treated with an effector according to claim 17.

20. The method according to claim 18 or 19, wherein the host is selected from microorganisms of the genus Ashbya.

21. The method according to any one of claims 18 to 20, wherein the microorganism is a host according to one of claims 10 to 12.

22. Use of a polynucleotide according to one of claims 1 to 4 and 6 or a polypeptide according to claim 7 as a target for modulating the production of vitamin B2 and / or precursors and / or derivatives thereof in a microorganism of the genus Ashbya.

23. Use of a polynucleotide according to one of claims 1 to 4 and 6 or a polypeptide according to claim 7 as a target for modulating the cell wall and / or cytoskeleton structure in a microorganism of the genus Ashbya during cultivation for the microbiological production of vitamin B2 and / or precursors and / or derivatives thereof.

24. Host according to claim 12 with a modified cell wall and or cytoskeleton.